Zwitterionic hydrogels for delivery of biomolecules

ABSTRACT

The invention provides a novel approach in which zwitterionic networks are used to sequester and deliver ionic biomolecules, such as proteins, without compromising their native conformation and bioactivity. Zwitterionic networks are designed to effectively retain and deliver ionic or polar biomolecules for guided tissue regeneration. The invention represents a conceptual advance and enables a novel strategy for the utilization of zwitterionic motifs as therapeutics delivery vehicles and tissue engineering scaffolds.

PRIORITY CLAIMS AND RELATED PATENT APPLICATIONS

This application claims the benefit of priority from U.S. ProvisionalApplication Ser. No. 62/022,187, filed on Jul. 8, 2014, the entirecontent of which is incorporated herein by reference in its entirety.

GOVERNMENT RIGHTS

This invention was made with Government support under grant no. AR055615awarded by the National Institutes of Health. The Government has certainrights in the invention.

TECHNICAL FIELD OF THE INVENTION

The invention generally relates to biomaterials, tissue engineering anddelivery of biomolecules. More particularly, the invention relates tozwitterionic polymers and methods of their applications in delivery ofbiomolecules and tissue engineering.

BACKGROUND OF THE INVENTION

Zwitterions, including phosphobetaine, sulfobetaine, and carboxybetaine,are well-known for their anti-biofouling properties as widelydemonstrated on 2-dimensional (2D) surfaces. The unique zwitterionicstructures, simultaneously possessing cationic and anionic residues yetoverall electronic neutral, exhibit strong affinity for water, therebygiving rise to super hydrophilic surfaces suppressing the hydrophobicinteractions known to denature proteins. (Lowe, et al. 2002 Chemicalreviews 102:4177-89; Seo, et al. 2008 Biomaterials 29:1367-76; Krishnan,et al. 2008 J Mater Chem 18:3405-13; Kane, et al. 2003 Langmuir19:2388-91.)

Zwitterionic motifs have also been shown to mimic the action of proteinstabilizing ions in stabilizing/maintaining the native conformation ofproteins and inhibiting non-specific protein adsorption, which is knownto set off undesired cascades of surface events (e.g., thrombosis,immune response). Accordingly, they have been largely exploited forconstructing anti-fouling surfaces/interfaces to inhibit protein,bacterial and cellular adhesions, and as bioinert implants for reducingscar tissue formation. (Nakaya, et al. 1999 Prog Polym Sci 24:143-81;Zhang, et al. 2003 Biomaterials 24:4223-31; Jiang, et al. 2010 Adv Mater22:920-32; Smith, et al. 2012 Sci Transl Med 4, 153; Ishihara, et al.1998 J Biomed Mater Res 39:323-30; Yuan, et al. 2003 Colloid Surface B29:247-56; Franz H. Zur Lehre von der Wirkung der Salze. Archiv fürExperimentelle Pathologie and Pharmakologie 1888;25; Han, et al. 2007Sci China Ser B 50:660-4; Keefe, et al. 2012 Nat Chem 4:60-4; Zhang, etal. 2013 Nat Biotechnol 31:553-6; Harris J M.Poly(ethyleneglycol)chemistry: biotechnical and biomedical applications.New York: Plenum Press; 1992; Horbett, et al. 1995 Proteins atInterfaces II: Fundamentals and Applications Washington, DC: Am. Chem.Soc.)

Recently, the use of zwitterionic sublfobetaine hydrogel to facilitatetemplated biomineralization was reported, which capitalizes on theability of the zwitterionic motifs to effectively recruit/nucleateoppositely charged mineralization precursor ions (e.g., Ca²⁺, PO₄ ³⁻)across the 3D hydrogel network. (Liu, et al. 2013 Biomaterials34:2442-54.)

There is little report, however, on whether 3-dimensionally presentedzwitterions can effectively sequester ionic biomolecules. Such aproperty, if intrinsically exists, could fundamentally change thecurrent perception of zwitterionic materials as being primarilyanti-biofouling and significantly broaden its potential use inbiomedical applications. It is strongly desired that novel methods andcompositions are uncovered and developed that greatly expand the utilityof zwitterionic materials in the bioengineering and therapeutics areas.

SUMMARY OF THE INVENTION

The invention provides a novel approach in which zwitterionic materialsare utilized to retain and deliver ionic biomolecules, such as proteins,for guided tissue regeneration. The invention uncovers and takesadvantage of the ability of zwitterionic networks to sequester ionicbiomacromolecules without compromising their native conformation andbioactivity, which challenges the conventional narrow perception andcategorization of zwitterionic materials as low-fouling and bioinert.The invention demonstrates that zwitterionic networks are versatilevehicles useful in engineering controlled bioactive microenvironment forbiomedical applications.

The invention represents a conceptual advance and enables a novelstrategy for the utilization of zwitterionic motifs as therapeuticsdelivery vehicles and tissue engineering scaffolds. The inventiondistinguishes zwitterionic materials from the current benchmarkbiocompatible and anti-fouling material poly(ethylene glycol) (PEG) thatis widely used in the biomaterials field. The ability of thezwitterionic hydrogel to promote the functional bone healing with anexceptionally low dose of therapeutic proteins, as demonstrated herein,can significantly reduce the cost as well as improve the safetyassociated with the protein therapeutics.

In one aspect, the invention generally relates to a composite materialcomprising a polymer network and a biologically active compound, whereinthe 3-dimensioanl polymer network comprises a zwitterionic moiety.

In another aspect, the invention generally relates to an implantcomprising a composite material characterized by a 3-dimensionalcrosslinked polymer network sequestered therein one or more biologicallyactive compounds, wherein the polymer comprises a zwitterionic moiety.

In yet another aspect, the invention generally relates to an implantcomprising a 3-dimensional scaffold comprising a 3-dimensioanl polymernetwork, wherein the polymer network comprises a zwitterionic moiety,adapted to sustained in vivo delivery of one or more biologically activecompounds.

In yet another aspect, the invention generally relates to an implantcomprising a n implant comprising a composite material characterized bya 3-dimensional crosslinked polymer network comprising a zwitterionicmoiety.

In yet another aspect, the invention generally relates to a method formaking a composite material useful for tissue engineering. The methodincludes crosslinking, in the presence of a biologically activecompound, a polymer comprising a zwitterionic moiety to form a3-dimensioanl crosslinked polymer network with the biologically activecompound encapsulated therein.

In yet another aspect, the invention generally relates to a method formaking a composite material useful for tissue engineering. The methodincludes: crosslinking a polymer comprising a zwitterionic moiety toform a 3-dimensioanl crosslinked polymer network; and contacting thecrosslinked polymer network with a solution of a biologically activecompound under conditions such that the biologically active compound issequestered in the crosslinked polymer network.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic illustrations of (a), the well-establishedanti-biofouling property of 2D zwitterionic surfaces vs (b),hypothesized protein-sequestering property of 3D zwitterionic networks.

FIG. 2. 3D zwitterionic hydrogel networks efficiently sequesteredrhBMP-2 and enabled its sustained in vitro release. (a), Sequestrationof rhBMP-2 by zwitterionic PSBMA vs non-ionic PEGMA control as afunction of crosslinker content (n=3, 0.33, 1.33 or 5.33 mol% relativeto monomers) after 2-h incubation in PBS. A 300-ng rhBMP-2 initialloading dose was applied to all hydrogels and the sequestered proteincontent was determined after 2-h incubation in PBS. (b), Swelling ratioby weight (S_(w)) of PSBMA vs PEGMA hydrogels (n=5) in PBS as a functionof crosslinker content (0.33, 1.33 or 5.33 mol% relative to monomers).(c), Swelling ratio by weight (S_(w)) of PSBMA vs PEGMA hydrogels (1.33mol% crosslinker content; n=5) in water and in PBS. (d), Cumulativerelease of the loaded 300-ng rhBMP-2 from three types of zwitterionichydrogels with identical crosslinker amount of 1.33 mol% (n=3). (e),Osteogenic trans-differentiation of C2C12 cells induced by the rhBMP-2sustained-released (between day 7 to day 9) from PSBMA vs PEGMAhydrogels as shown by the expression of osteogenic marker ALP (redstains). C2C12 culture directly supplemented with 300-ng rhBMP-2 withoutany hydrogel carrier served as a positive control.*p<0.05 (two-wayANOVA).

FIG. 3. High-efficient in vivo local delivery of rhBMP-2 by PSBMAhydrogel implant as examined by the 5-mm rat femoral segmental defectmodel. (a), A PSBMA hydrogel implant (5 mm×3 mm×3 mm) with/withoutrhBMP-2 press-fit within the femoral segmental defect stabilized by aradiolucent polyetheretherketone (PEEK) plate fixator. (b),Reconstructed μ-CT 3D images & 2D bone mineral density color mapping ofthe center longitudinal slice of the defect treated with PSBMA hydrogelgrafts with/without 500-ng rhBMP-2 at 4 and 12 weeks post-op. (c), Bonevolume & (d), Bone mineral density of the defects (n=4) treated withPSBMA hydrogel grafts with/ without 500-ng rhBMP-2 at 4, 8 and 12 weekspost-op. *p<0.05 (two-way ANOVA) (e), Peak torque of the 12-weekexplants treated with PSBMA hydrogel grafts with/without rhBMP-2 (n=3)vs age-matched intact femurs (n=6). *p<0.05 (Student's T-test). (f),Reconstructed μ-CT 3D image & 2D bone mineral density color mapping ofthe center transverse slice of the defect treated with PSBMA hydrogelgraft with 500-ng rhBMP-2 at 12 weeks post-op showing mature bony callusfully encapsulating the rhBMP-2 loaded PSBMA hydrogel scaffold. (g), H&Estaining of the 12-week explant showing robust new bone (NB) fullyencapsulating the rhBMP-2 loaded PSBMA scaffold and integrated withadjacent native cortical bone (CB). BM=bone marrow. Black arrows in theenlarged image denote hydrogel scaffolds integrated with the NB.

FIG. 4. Temporally sequestered rhBMP-2 increased the cell attachment &ECM deposition on the low-fouling zwitterionic PSBMA hydrogel implant.(a), Confocal images of in vivo endogenous cell attachment on thesurface of PSBMA explants with/without rhBMP-2 at day 2 and 7 post-op.Actin was stained by Alexa phalloidin (red) while nuclei were stained byDAPI (blue). (b), H&E staining of the ECM deposition on the explantswith/without rhBMP-2 at day 2 and 7 post-op.

FIG. 5. Mineralization outcomes of zwitterionic pSBMA vs non-ionic pHEMAhydrogels as examined by SEM and CT. All hydrogels were crosslinked by1.33 mol% of EGDMA relative to monomers. The hydrogels were placed in anaqueous acidic solution of hydroxyapaptite (pH=2.5-3.0, 14.7 mg/mL)containing 2-M urea, and subjected to controlled heating from 37 ° C. to95 ° C. at 0.2 ° C/min. In the absence of ionic motifs, themineralization of the non-ionic pHEMA hydrogel occurred exclusively onthe surface. With both positive and negative charged residuesfacilitating the penetration of oppositely charged mineralizationprecursor ions (e.g. Ca²⁺, PO₄ ³⁻) across the hydrogel, the zwitterionicpSBMA templated extensive mineralization throughout the 3D hydrogel.

FIG. 6. Schematic illustration of the preparation of hydrogels from PEGand zwitterionic methacrylate monomers, and the loading of rhBMP-2solutions on hydrogels though the de-swelling/swelling process. Allhydrogels were prepared with identical crosslinker content of 1.33 mol%relative to monomers.

FIG. 7. Cumulative in vitro release of rhBMP-2 from (a) zwitterionicPSBMA and (b) non-ionic PEGMA hydrogels (n=3) as a function of PEGDMAcrosslinker content in PBS (pH 7.4) as determined by the BMP-2Quantikine kit (R&D Systems). Initial rhBMP-2 loading dose: 300ng/hydrogel (cylindrical) specimen.

FIG. 8. Free water fraction (R_(ƒ)) of PSBMA vs PEGMA hydrogels (n=3)equilibrated in PBS as determined by DSC. The difference between the twogroups is significant (p<0.05, Student's T-test).

FIG. 9. Radiographic monitoring over time of the bony callus formationover the 5-mm rat femoral segmental defects treated with PSBMA hydrogelgrafts with/without 500-ng rhBMP-2.

FIG. 10. Reconstructed μ-CT 3D images, longitudinal cross-section views,and longitudinal 2D bone mineral density color mapping of the 5-mmfemoral segmental defect treated with PSBMA hydrogel grafts with 500-ngrhBMP-2 over time.

FIG. 11. Reconstructed μ-CT 3D images, longitudinal cross-section views,and longitudinal 2D bone mineral density color mapping of the 5-mmfemoral segmental defect treated with PSBMA hydrogel grafts alone overtime.

FIG. 12. Reconstructed μ-CT 3D images of all 5-mm femoral segmentaldefects treated with PSBMA hydrogel implants with/without the loading of500-ng rhBMP-2 at 12 weeks post-op.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel approach in which zwitterionic networksare used to sequester and deliver ionic biomolecules, such as proteins,without compromising their native conformation and bioactivity.Zwitterionic networks are designed to effectively retain and deliverionic or polar biomolecules for guided tissue regeneration. Theinvention represents a conceptual advance and enables a novel strategyfor the utilization of zwitterionic motifs as therapeutics deliveryvehicles and tissue engineering scaffolds.

In contrast to the conventional narrow perception and categorization ofzwitterionic materials as low-fouling and bioinert, the inventiongreatly expands the utilities of zwitterionic materials in thebioengineering and therapeutic areas. Zwitterionic networks aredemonstrated as effective and versatile vehicles for engineeringcontrolled bioactive microenvironment for biomedical applications. Theinvention distinguishes zwitterionic materials from the currentbenchmark biocompatible and anti-fouling material poly(ethylene glycol)(PEG) that is widely used in the biomaterials field. As demonstratedherein, the ability of the zwitterionic hydrogel to promote thefunctional bone healing with an exceptionally low dose of therapeuticproteins can significantly reduce the cost and improve the safetyassociated with the protein therapeutics.

For example, as disclosed herein, 3-dimensionally (3D) presentedzwitterionic motifs (e.g., in crosslinked hydrogels), effectivelysequestered osteogenic bone morphogenetic protein-2 (rhBMP-2). The ionicinteractions between rhBMP-2 and the 3D zwitterionic network enableddynamic sequestering and sustained release of the protein with preservedbioactivity. The zwitterionic hydrogel allowed high-efficiency in vivolocal delivery of rhBMP-2, which can template the functional healing ofcritical-size femoral segmental defects in rats with rhBMP-2 at aloading dose substantially lower than those required for current naturalor synthetic polymeric carriers. The sequestered rhBMP-2 can besustainedly released well over a week with well-preserved bioactivity,driven by the dynamic ionic interactions of rhBMP-2 with the3-dimensionally presented zwitterionic motifs rather than by scaffoldbiodegradations.

Such sequestration and high-efficiency delivery of rhBMP-2 allowedrobust repair of critical-size rat femoral segmental defects templatedby the zwitterionic hydrogel implant at an exceptionally low loadingdose of 500-ng rhBMP-2.

Zwitterions (e.g., phosphobetaine, sulfobetaine, and carboxybetaine) arewell known for their anti-biofouling properties as widely demonstratedon 2-dimensional (2D) surfaces (FIG. 1a ). The unique zwitterionicstructures, simultaneously possessing cationic and anionic residues yetoverall electronic neutral, exhibit strong affinity for water, therebygiving rise to super hydrophilic surfaces suppressing the hydrophobicinteractions known to denature proteins. Zwitterionic sublfobetainehydrogel have been reported to facilitate templated biomineralizationwas reported, which capitalizes on the ability of the zwitterionicmotifs to effectively recruit/nucleate oppositely charged mineralizationprecursor ions (e.g., Ca²⁺, PO₄ ³⁻) across the 3D hydrogel network.(Liu, et al. 2013 Biomaterials 34:2442-54.) Unlike non-ionic hydrogelthat was only able to template the mineralization on the surface, thezwitterionic hydrogel enabled extensive mineralization throughout the 3Dnetwork, supporting the critical role of zwitterionic motifs inrecruiting precursor ions (FIG. 5). Prior to the disclosure herein,there has been no report on whether 3D zwitterionic motifs caneffectively sequester ionic biomacromolecules, such as protein, theireffective retention and sustained release (FIG. 1b ).

Effective Sequestration of Proteins by 3D Zwitterionic Hydrogels

As examples, simple crosslinked polymethacrylate hydrogels bearingzwitterionic side chains were prepared. The in vitrosequestration/release profile of osteogenic human recombinant bonemorphogenetic protein-2 (rhBMP-2) from the zwitterionic hydrogels wasinvestigated and compared with that of the non-ionic low-foulingpoly(ethylene glycol) hydrogel control. The efficacy of a zwitterionicsulfobetaine hydrogel in delivering rhBMP-2 in vivo to promote thefunctional healing of critical-size (5-mm) femoral segmental defects inrats and endogenous cell attachment on the otherwise low-fouling implantwas investigated.

The zwitterionic PSBMA hydrogels were prepared by photo-crosslinkingsulfobetaine methacrylate (SBMA) with varying contents of crosslinkerPEGDMA. And a poly(ethylene glycol) methacrylate (PEGMA) hydrogelsbearing non-ionic poly(ethylene glycol) (PEG), another well-establishedanti-biofouling motif, were prepared at the identical crosslinkercontents as controls (FIG. 6 & Table 1). To examine the efficiency ofthe hydrogels for sequestering therapeutic proteins, 300 ng of rhBMP-2(in 10 μL PBS solution) was loaded on each partially dried hydrogel andallowed to equilibrate at 37 ° C. for 1 h to ensure complete absorptionof the aqueous solution (FIG. 6).

TABLE 1 Formulations of the photo-crosslinked hydrogels with identicalcrosslinker content VA-086 Monomer Crosslinker stock amount amount^(a)solution^(b) PBS Monomer (mmol) (μL) (μL) (μL) SBMA 2 17.9 100 1882.1PEGMA 2 17.9 100 1882.1 MPC 2 17.9 100 1882.1 CBMA 2 17.9 100 1882.1^(a)PEGDMA (M_(n) = 750). ^(b)2% (w/v) VA-086 in PBS.

Although zwitterionic sulfobetaine and PEG surfaces are both known forresisting non-specific protein absorptions, the respective 3D networksexhibited significant differences in sequestering rhBMP-2 even with asimilar swelling ratio at the identical crosslinker content of 5.33 mol%(relative to monomer; FIGS. 2a & 2 b). The non-ionic PEGMA hydrogelcould not effectively sequester rhBMP-2, with only about 10% of theinitially loaded rhBMP-2 retained on the hydrogel after 2-h incubationin PBS (FIG. 2a ). This observation is consistent with previous findingsthat PEG hydrogels lack affinity for ionic proteins. (Zhu 2010Biomaterials 31:4639-56; Place, et al. 2009 Nature Materials 8:457-70.)By contrast, about 60% of the initially loaded rhBMP-2 was sequesteredby the zwitterionic PSBMA network of the same crosslinker content (5.33mol%) after 2-h incubation (FIG. 2a ). Given the similar swelling ratio,thus similar diffusibility of solutes across the 3D network, thedifferent efficiencies of sequestering ionic proteins observed with thetwo identically crosslinked networks was likely due to the differentionic states of their side chains. (Hoffman 2002 Advanced drug deliveryreviews 54:3-12.)

By reducing the degree of chemical crosslinking by up to 16-fold, it wasshown that the zwitterionic PSBMA network swelled significantly in PBSby up to 10-fold while no significant crosslinker content-dependentchanges in swelling ratio in PBS was observed with the non-ionic PEGMAnetwork (FIG. 2b ). This further supported that the different ionicstates of side chains presented in the two 3D networks (zwitterionic vsnon-ionic) can translate into significant differences in their physicalproperties in ionic environment, including different swelling behaviorand efficiencies in sequestering/releasing ionic protein (FIGS. 2a and7).

Furthermore, unlike the non-ionic PEGMA network that was insensitive tothe presence of salts (no significant difference in swelling ratios inwater vs in PBS, pH 7.4, FIG. 2c ), the zwitterionic PSBMA networkexpanded almost 400% more in PBS than in water (FIG. 2b ). Such anantipolyelectrolyte swelling behavior can be attributed to thedisruption of the intermolecular salt bridges formed between the anionicsulfonate and cationic ammonium residues by ionic solutes. (Poynton, etal. 2002 Spine 27:S40-S8.) Combined with the higher free water fractionsin the equilibrated zwitterionic PSBMA hydrogel (85% in PSBMA vs 69% inPEGMA, FIG. 8), this observation further supports that theionic-sensitive nature of the zwitterionic network is beneficial to thediffusion of ionic solutes in general across the 3D network.

Taken together, these data validate that ionic interactions play anindispensable role in effectively sequestering rhBMP-2 by thezwitterionic PSBMA network. Similar rhBMP-2 retention profiles were alsoobserved with the 3D zwitterionic networks bearing phosphobetaine (PMPC)and carboxybetaine (PCBMA) motifs (FIG. 2d ), supporting effectiveprotein retention as a novel yet generalizable feature for 3Dzwitterionic matrices.

Sustained Release of Bioactive Proteins from Zwitterionic Hydrogels

Monitoring of the rhBMP-2 release from the hydrogels within the first 24h of incubation in PBS by ELISA revealed ˜30% release of the initiallyloaded protein in the first 2 h, followed by a 3% of slower release inthe next 22 h (FIG. 2d ), leaving >65% sequestered by the zwitterionicPSBMA (1.33 mol% crosslinker content) by 24 h.

To examine whether the rhBMP-2 sequested by the PSBMA hydrogel could becontinually released with retained bioactivity over a much longer periodof time, an established culture model of BMP-2-induced osteogenictrans-differentiation of murine myoblast C2C12 cells was used.(Katagiri, et al. 1994 J Cell Biol 127:1755-66; Liu, et al. 2011 ActaBiomaterialia 7:3488-95.) This model was chosen over BMP-2-inducedosteogenesis of mesenchymal stem cells (MSCs) due to the complete lackof expression of osteogneic markers by C2C12 cells prior to BMP-2induction (thus much cleaner background than MSCs). It was shown thatwhen the rhBMP-2-bearing PSBMA was placed in murine myoblast C2C12cutlure after a 6-day pre-incubation in PBS, the further sustainaedlyreleased rhBMP-2 (from day 7 to day 9) from the PSBMA hydrogel was ableto induce robust osteogenic trans-differentiation of C2C12 cells intoalkaline phosphatase (ALP)-expressing osteoblasts (FIG. 2e ). Theintense ALP staining, comparable to that observed with the positivecontrol culture (FIG. 2e ) where 300-ng rhBMP-2 was directlysupplemented without any carrier, suggest that the bioactivity of thesequestered and subsequently released rhBMP-2 was well preserved for atleast 9 days.

This result is in stark contrast to the minimal ALP stains detected fromthe C2C12 culture supplemented with the PEGMA hydrogel subjected toidentical BMP-2 loading and PBS pre-incubation treatment, consistentwith the much poorer initial sequestration of rhBMP-2 by the non-ionicPEGMA hydrogel. It is worth noting that the circulation half-life ofrhBMP-2 and most other growth factors, when in free form, tends to bevery limited (e.g., 7-16 min for rhBMP-2). (Poynton, et al. 2002 Spine27:S40-S8.) Here, well-preserved bioactivity of the rhBMP-2 sequesteredby the PSBMA hydrogel was demonstrated well over a week. This may beattributed to the superhydrophilic structrual water surroundingzwitterioic residues that prevent protein denaturing and the Hofmeisterions-like effect of the zwitterions for stabilizing native proteinconformations. (Kane, et al. 2003 Langmuir 19:2388-91; Franz H. ZurLehre von der Wirkung der Salze. Archiv für Experimentelle Pathologieand Pharmakologie 1888;25; Keefe, et al. 2012 Nat Chem 4:60-4.) Overall,these observations support the zwitterionic PSBMA hydrogel as aneffective carrier for the high-efficiency sequestration and sustainedlong-term release of theapeutic proteins such as rhBMP-2.

Treating Bone Defects by High-Efficiency In Vivo Delivery of Proteins

To test the in vivo efficacy of the PSBMA hydrogel as a syntheticimplant with rhBMP-2 delivery capability, the repair of 5-mm rat femoralsegmental defect, an established critical-size non-union model,templated by the PSBMA implant with or without pre-loaded rhBMP-2 wasevaluated (FIG. 3a ). (Filion, et al. 2011 Tissue Eng Pt A 17:503-11;Uhrig, et al. 2013 Bone 55:410-7.)

Current clinical use of rhBMP-2, delivered via absorbable collagensponge carrier) (INFUSE®), to stimulate spine fusion or tibial fracturerepair require exceedingly high loading doses comparable to ˜1.5 mg permilliliter volume of defect (1500 ng/mm³). Such a supra-physiologicaldosages and their burst release from the sub-optimal collagen carrierhave resulted in significant systemic and local adverse effects. Loadingdoses ranging from 2 to 50-μg rhBMP-2/scaffold (˜250 to 6,250ng/mm-defect) have been typically used to achieve adequate repair ofcritical-size long bone or trabecular bone defects in rats with eithernatural or synthetic polymeric carriers. Table 2 lists representativereported rhBMP-2 loading doses on various natural or synthetic polymericscaffolds used for achieving adequate healing of critical-size bonedefects in rats. Literatures reporting synergistic loading of rhBMP-2along with other growth factors/therapeutics are not included.

TABLE 2 Representative literature rhBMP-2 loading doses on variousnatural or synthetic polymeric scaffolds rhBMP-2 loading dose μg/mm-Scaffold type Scaffold materials Defect model μg/scaffold defect μg/mm³Natural Collagen 1.5 (INFUSE ®)^(a) Gelatin 6-mm segmental, ulna 3 0.5Alginate 8-mm segmental, femur 2 0.25 5 0.63 Keratose 8-mm segmental,femur 50 6.25 Silk 5-mm segmental, femur 2.5 0.5 Hyaluronic acid 5-mmcranium 5 Synthetic PPF/TCP 5-mm segmental, femur 10 2 PLGA & PPF 5-mmsegmental, femur 6.5 1.3 PLA-DX-PEG 4-mm, ilia 10 PEG-RGD 8-mm, cranium5 PSBMA^(b) 5-mm segmental, femur 0.5 0.1 0.01 ^(a)commercial rhBMP-2delivery scaffolds approved by FDA. ^(b)zwitterionic PSBMA hydrogelscaffold used in the current study. ^(c)References: Mckay, et al. 2007Int’l orthopaedics 31:729-34; Carragee, et al. 2011 Spine J 11:471-91;Ratanavaraporn, et al. 2011 Biomaterials 32:2797-811; Kolambkar, et al.2011 Bone 49:485-92; de Guzman, et al. 2013 Biomaterials 34:1644-56;Kirker-Head, et al. 2007 Bone 41:247-55; Patterson, et al. 2010Biomaterials 31:6772-81; Kempen, et al. 2009 Biomaterials 30:2816-25;Lutolf, et al. 2003 Nat Biotechnol 21:513-8; Chu, et al. 2007Biomaterials 28:459-67; et al. 2001 NatBiotechnol 19:332-5.

Loading doses of rhBMP-2 less than 2 μg (without synergistic delivery ofother growth factor therapeutics) often resulted ininadequate/inconsistent repair outcomes. (Schmoekel, et al. 2005Biotech. & bioeng. 89:253-62.) Here, in contrast, a significantly lowerloading dose of 500-ng rhBMP-2 was applied to the PSBMA scaffold(equivalent to ˜11 ng/mm³ or 100 ng/mm-defect) press-fit into the 5-mmrat femoral segmental defect. It is belived that consistent functionalhealing of critical rat long bone defect with such a low loading dose ofrhBMP-2 alone has never been reported before.

At 2 weeks, mineralized healing callus emerged around the defectsimplanted with PSBMA with rhBMP-2 (FIG. 9). Strikingly, the bony callusstarted to bridge over the defect by as early as 4 weeks (FIG. 3b , FIG.10), and by 12 weeks, mature and uniform bony callus characterized withrecanalization and high bone mineral density (FIGS. 3b, 3d, 3f , FIG.10) fully encapsulated the defect, leading to substantial restoration(˜40% compared to intact age matched femur control) of the torsionalrigidity of the defect (FIG. 3e ).

Continued remodeling of the new bone is expected to further increase thetorsional rigidity over time. In the absence of rhBMP-2, the PSBMA alsoled to the early onset (FIG. 9) and steady growth of bony callus overthe course of 12 weeks as characterized with increasing bone volume(FIG. 3c ) and bone mineral density (FIG. 3d ). However, in the absenceof rhBMP-2, the calcified callus failed to bridge over the entiredefects by 12 weeks (FIGS. 3b , 9, 11 & 12) to restore the biomechanicalintegrity of the defect (FIG. 3e ). Of note, although fairly high bonevolumes were detected at the regions of interest (ROI) in both treatmentgroups by 12 weeks (FIG. 3c , no statistically significant difference),the rhBMP-2 treated group consistently guided uniform bony callusformation across the full length of the defect whereas the new boneformation templated by the no-BMP-2 control group was primarilylocalized around the graft-cortical bone junctions (FIG. 12).

Transverse cross-sectional view of the repaired defect (FIG. 3f ) andH&E staining of the explant at 12-week post-op (FIG. 3g ) revealed thatthe bony callus formation was tightly templated by and integrated withthe PSBMA hydrogel (note that the disintegration/shrinkage of hydrogelscaffold trapped within the bony callus was a histology processingartifact as the hydrogel shrank dramatically upon dehydration).

These data supported that PSBMA hydrogel implant is a highly effectivecarrier for the local delivery of rhBMP-2, which enabled the functionalrepair of rat critical-size long bone defect at a significantly reducedBMP-2 loading dose that is desired from both safety andcost-effectiveness perspectives.

rhBMP-2 Sequestration Promoting Endogenous Cell Attachment & ECMDeposition on the Otherwise Low-Fouling Surface of Zwitterionic PSBMAHydrogel Implant

The robust early bone healing enabled by PSBMA in the presence ofrhBMP-2 across the entire defect suggests that a cascade of cellularevents required for initiating bone healing must have occurred in atimely manner along the implant surface, counterintuitive to theperception that zwitterionic surfaces and scaffolds tend to reduceprotein absorptions/cellular adhesion. (Smith, et al. 2012 Sci TranslMed 4, 153; Zhang, et al. 2013 Nat Biotechnol 31:553-6; Bose, et al.2012 Trends in biotechnology 30:546-54; Liu, et al. 2009 Biomacromol.10:2809-16.)

Also investigated was the early stage in vivo cell attachment during theguided bone healing with and without the loading of rhBMP-2. The resultsshowed that the retention of rhBMP-2 by the PSBMA hydrogel implantsshifted the microenvironment of the zwitterionic scaffolds fromlow-fouling to cell adhesive. As revealed by fluorescent microscopy andH&E staining, only limited cell attachment was observed on the surfaceof the PSBMA hydrogel without rhBMP-2 within the first 2 dayspost-implantation with no obvious increases by 7 days (FIG. 4). This isconsistent with the low-fouling nature of zwitterionic surfaces as wellas the recent report that zwitterionic carboxybetaine hydrogelssuppressed fibrous tissue encapsulation in vivo. (Zhang, et al. 2013 NatBiotechnol 31:553-6)

In contrast, substantially more endogenous cells attached to the surfaceof the rhBMP-2-bearing PSBMA implant at 2 days post-implantation (FIG.4), and these adherent cells continued to proliferate and led to moreeffective ECM deposition, and presumably the initiation of callusformation, at day 7 post-implantation. These observations suggest thatthe ionic retention of rhBMP-2 on the 3D zwitterionic scaffold not onlyintroduced osteoinductivity, but also improved the osteoconductivity ofthe otherwise commonly perceived low-fouling and bioinert scaffold,enabling facile cellular attachment. As many ECM components such asfibronectin, collagen and laminin have high affinity for heparin-bindinggrowth factors like BMPs, the rhBMP-2-bearing scaffold in turn couldfacilitate the attachment of these ECM components and subsequentcellular adhesion and more uniform and robust bony callus formation.(Ruoslahti, et al. 1991 Cell 64:867-9.)

Thus, in one aspect, the invention generally relates to a compositematerial comprising a polymer network and a biologically activecompound, wherein the 3-dimensioanl polymer network comprises azwitterionic moiety.

In certain embodiments, the biologically active compound is abiomacromolecule. In certain embodiments, the biologically activecompound is a small molecule compound. In certain embodiments, thebiologically active compound is a biomacromolecule such as an ionic orpolar protein or peptide.

In certain preferred embodiments, the biomacromolecule is a therapeuticosteogenic protein, an anabolic agent or any angiogenic factor, forexample, selected from BMPs (e.g., rhBMP-2, rhBMP-7, rhBMP-2/7heterodimer), TGF-beta, EGF, FGF, IGF-1, and VEGF. In certainembodiments, the therapeutic osteogenic protein is present in thepolymer network at a loading from about 1 ng to about 20,000 ng (e.g.,from about 1 ng to about 20,000 ng, from about 10 ng to about 20,000 ng,from about 100 ng to about 20,000 ng, from about 1,000 ng to about20,000 ng, from about 5,000 ng to about 20,000 ng, from about 10,000 ngto about 20,000 ng, from about 1 ng to about 10,000 ng, from about 1 ngto about 5,000 ng, from about 1 ng to about 3,000 ng, from about 1 ng toabout 1,000 ng, from about 1 ng to about 500 ng, from about 1 ng toabout 300 ng, from about 1 ng to about 100 ng) per critical-size femoralsegmental defect (e.g., in rat or scaled to human defect sizesaccordingly).

The polymer network is preferably a 3-dimensioanl crosslinked polymernetwork. Any suitable polymer network may be utilized, for example, acrosslinked hydrogel of polymethacrylate, polyacrylate,polymethacrylamide or polyacrylamide. Any suitable crosslinkers may beutilized, for example, selected from poly(ethylene glycol)dimethacrylate, poly(ethylene glycol) diacrylate, ethylene glycoldiacrylate and ethylene glycol dimethacrylate, or derivatives thereof(e.g., amides). The polymer network may be crosslinked to any suitablecrosslinking density, for example, from about 0.05 mol% to about 10 mol%(e.g., from about 0.05 mol% to about 5 mol%, from about 0.05 mol% toabout 3 mol%, from about 0.05 mol% to about 1 mol%, from about 0.05 mol%to about 0.5 mol%, from about 0.05 mol% to about 0.1 mol%, from about0.1 mol% to about 10 mol%, from about 0.5 mol% to about 10 mol%, fromabout 1 mol% to about 10 mol%, from about 5 mol% to about 10 mol%).

Any suitable zwitterionic moieties may be incorporated in the polymernetwork, for example, one or more selected from sulfobetaine,phosphorylcholine and carboxybetaine. The zwitterionic moieties may bepresent in a polymer network in the backbone and/or as pendant groups toa polymeric backbone. The zwitterionic moieties may be present in thepolymer network at any suituable density, for example, from about 1 mol%to about 100 mol% (e.g., from about 1 mol% to about 50 mol%, from about1 mol% to about 30 mol%, from about 1 mol% to about 30 mol%, from about1 mol% to about 10 mol%, from about 1 mol% to about 5 mol%, from about 5mol% to about 100 mol%, from about 10 mol% to about 100 mol%, from about30 mol% to about 100 mol%, from about 50 mol% to about 100 mol%).

In certain embodiments, the composite material is biodegradable.

In another aspect, the invention generally relates to an implantcomprising a composite material characterized by a 3-dimensionalcrosslinked polymer network sequestered therein one or more biologicallyactive compounds, wherein the polymer comprises a zwitterionic moiety.

In yet another aspect, the invention generally relates to an implantcomprising a 3-dimensional scaffold comprising a 3-dimensioanl polymernetwork, wherein the polymer network comprises a zwitterionic moiety,adapted to sustained in vivo delivery of one or more biologically activecompounds.

In yet another aspect, the invention generally relates to an implantcomprising a n implant comprising a composite material characterized bya 3-dimensional crosslinked polymer network comprising a zwitterionicmoiety.

In certain preferred embodiments, the implant of the invention issuitable for treating dental, bone, cartilage, tendon, ligament orosteochondral damage.

In yet another aspect, the invention generally relates to a method formaking a composite material useful for tissue engineering. The methodincludes crosslinking, in the presence of a biologically activecompound, a polymer comprising a zwitterionic moiety to form a3-dimensioanl crosslinked polymer network with the biologically activecompound encapsulated therein.

In yet another aspect, the invention generally relates to a method formaking a composite material useful for tissue engineering. The methodincludes: crosslinking a polymer comprising a zwitterionic moiety toform a 3-dimensioanl crosslinked polymer network; and contacting thecrosslinked polymer network with a solution of a biologically activecompound under conditions such that the biologically active compound issequestered in the crosslinked polymer network.

EXPERIMENTAL

Preparation of Hydrogels

Zwitterionic hydrogelspoly[2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide(PSBMA), poly(2-Methacryloyloxyethyl phosphorylcholine) (PMPC),poly[3-((2-(methacryloyloxy)ethyl)dimethylammonio)propanoate] (PCBMA)and nonionic poly(ethylene glycol) methacrylate (PEGMA, Mn=360) wereprepared (Table 1). Monomers SBMA, MPC and PEGMA (Mn=360) andcrosslinker poly (ethylene glycol) dimethacrylate (PEGDMA, Mn=750) werepurchased from Aldrich (St. Louis, MO), while CBMA was synthesized asreported. (Zhang, et al. 2006 Langmuir 22:10072-7.) The radicalinhibitors in PEGMA and PEGDMA were removed by passing through analuminum oxide column prior to use. In a typical procedure, 2 mmolrespective monomer was combined with 17.9 μL of PEGDMA, 100 μL of PBSsolution of 2,2′-Azobis[2-methyl-N-(2-hydroxyethyl)propionamide](VA-086, 2%, w/v), and 1882.1 μL of PBS. The mixture was bath-sonicated,and sterilized by passing through 0.22-μm polyethersulfone (PES)membrane filter (Millipore). The resulting solution was transferred to acustom-made Teflon mold with cylindrical (6 mm in diameter, 50 μL/well),square prism (5 mm×5 mm, 50 μL/well) or rectangle (6.5×32.6 mm, 400μL/well) wells and solidified under the irradiation of 365-nm light for10 min in a sterile hood. The hydrogels were stored in sterile PBS untilfurther uses.

Swelling Ratios of the Hydrogels

The swelling ratios by weight (Sw) of the hydrogels were determined inMilli-Q water or in PBS (pH=7.4) at room temperature according toEquation 1:

$\begin{matrix}{S_{W} = \frac{W_{h} - W_{d}}{W_{h}}} & {{Eq} - 1}\end{matrix}$

where W_(h) and W_(d) are the weight of the hydrogel in fully hydratedstate in water/PBS and freeze-dried state, respectively.

Free Water Fraction in the Hydrogels

The free water fraction in the hydrogels was measured by differentialscanning calorimetry (DSC) on a Q200 Modulated DSC (TA Instruments).About 15 mg of hydrogel equilibrated either in water or PBS was placedin an aluminum pan. The pan was then sealed tightly to prevent waterevaporation during the measurement. The testing was carried out from-40° C. to 40° C. at a heating rate of 2° C./min. The exothermal peakaround 0° C., attributed to the melting of the free water[19], wascalculated as ΔH_(endo), and the free water fraction (R_(ƒ)) within thehydrogel was determined according Equation 2:

$\begin{matrix}{R_{f} = \frac{\Delta H_{endo}}{\Delta H_{w}}} & {{Eq} - 2}\end{matrix}$

where ΔH_(w) is the heat fusion of pure water (332.2 mJ/mg)[6].

In Vitro Retention and Sustained Release of rhBMP-2

Recombinant protein rhBMP-2 (R&D Systems, CHO-derived) was reconstitutedaccording to vendor specifications and diluted with Ca²⁺/Mg²⁺-freeDulbecco's phosphate-buffered saline (DPBS, pH 7.4) to a loadingconcentration of 30 ng/μL. Hydrogels retrieved from the sterile stocksolution were partially dried in a sterile cell culture hood (with a gelvolume reduction of 50 to 100 mm³), and then transferred into the wellsof ultra-low attachment 24-well plate (Corning). Reconstituted rhBMP-2solution (10 μL, 30 ng/ μL) was placed on each hydrogel to achieve atotal loading dose of 300-ng rhBMP-2/hydrogel (cylindrical), and allowedto be incubated at 37° C. for 1 h (during which rhBMP-2 solutions werefully absorbed by the hydrogels). The rhBMP-2loaded hydrogels were thenincubated in 1 mL of DPBS at 37° C. for 2, 4, 6, 10, and 24 h.Concentration of the released rhBMP-2 in the DPBS at various time pointswere determined by an enzyme-linked immuno sorbent assay (ELISA) using arhBMP-2 Quantikine Kit (R&D Systems) and the amount of the rhBMP-2released form hydrogels were calculated from the standard curvegenerated during the same experiment. A sample size of 3 was applied toeach hydrogel group.

Bioactivity of the rhBMP-2 Sequestered on & Released from the Hydrogels

The bioactivity of the rhBMP-2 retained on and subsequently releasedfrom the PEGMA and PSBMA hydrogels was evaluated by their ability toinduce osteogenic trans-differentiation of murine myoblast C2C12 cellsinto osteoblasts. (Liu, et al. 2011 Acta Biomaterialia 7:3488-95;Filion, et al. 2011 Tissue Engineering Part A 17:503-11.) C2C12 cellswere seeded on 24-well cell culture plate (10,000 cells/cm²) in 1 mL ofDulbecco's modified eagle medium (DMEM) supplemented with 10% fetalbovine serum and 1% penicillin/streptomycin, and allowed to attachovernight. The medium was then replaced with fresh DMEM supplementedwith 5% fetal bovine serum and 1% penicillin/streptomycin, and therhBMP-2 loaded hydrogels retrieved from prior incubation in PBS up to 6days were placed in the adherent C2C12 culture. After 3 days, thehydrogel was removed and the cells were fixed and stained for alkalinephosphatase (ALP) using a Sigma Leukocyte Alkaline Phosphatase Kitaccording to the vender's protocol. C2C12 culture directly supplementedwith 300-ng rhBMP-2 without any hydrogel carrier served as a positivecontrol.

Animal Surgical Procedures

All animal procedures were approved by the University of MassachusettsMedical School Institutional Animal Care and Use Committee. Briefly,male Charles River SASCO-SD rats (289-300 g) were sedated and maintainedby 2% isoflurane—oxygen throughout the surgery. The mid-shaft of a femurwas exposed by a combination of sharp and blunt dissections and theperiosteum of the exposed femur was circumferentially removed to emulatea challenging clinical scenario where this important source ofprogenitor cells and signaling molecules is lost. (Filion, et al. 2011Tissue Engineering Part A 17:503-11.) A radiolucent, weight bearingpolyetheretherketone (PEEK) internal fixation plate was secured to theexposed femur with four bicortical screws into predrilled holes. A 5-mmmid-diaphyseal defect was then created using an oscillating Hall sawwith parallel blades. The defect site was thoroughly irrigated withsaline to remove bone debris and residue detached periosteum before itwas press-fit with a hydrogel graft with or without 500-ng rhBMP-2 (FIG.3a , n=4). The muscle and skin were closed with resorbable sutures andthe rats were given cefazolin (20 mg/kg, once a day) and bupenorphine(0.08 mg/kg, 3 times a day) injections subcutaneously over the next 2days. Rats were radiographed biweekly post-op to ensure proper graftpositioning, and subjected to monthly longitudinal microCT (μ-CT) scans(n=4) to quantitatively monitor the mineralized callus formation untiltime of sacrifice at 12 weeks post-op. For end-time point analyses, thefemur, with the PEEK plate fixator intact, was carefully separated fromthe adjacent hip and knee joints for either torsion test (n=3) orhistological staining. In a second set of experiments, implants wereretrieved at 2 and 7 days post-op (n=2) for examination of cellularattachment on the surface of the implant.

Longitudinal μ-CT Analysis

Rats were scanned immediately post-op and every 4 weeks thereafter on aviva-CT 75 in vivo Micro-CT system (SCANCO Medical AG) to monitor newbone formation over time. The effective voxel size of the reconstructedimages was 30×30×30 μm³. Data were globally thresholded and 3D images ofthe 5-mm defect, defined as the region of interest (ROI, 167 slices, 30μm / slice), were reconstructed for quantification of bone volume (BV,mm³) and bone mineral density (BMD, mgHA/ccm). Two-dimensional (2D)mineral density color mapping was generated by reconstructing therespective AIM file with a colored density gradient range of 1.5-3.5(1/mm). An unimplanted PSBMA hydrogel was scanned to guide propersetting of the threshold (to eliminate hydrogel background) for allanalyses.

Torsion Test

Explanted femora were torqued to failure as previously described toassess the degree of the functional restoration of their biomechanicalintegrity. (Filion, et al. 2011 Tissue Eng Pt A 17:503-11.) Briefly,explant was potted in stainless steel hexanuts with poly(methylmethacrylate). The PEEK plate fixators were either carefully bisectedwithout disturbing the underlying graft/new bone using a high-speed burr(the PSBMA group) or unscrewed and removed from the explants (rhBMP-2treated group) before mounted on the mini-torsion tester (ADMET Inc.).Each specimen was torqued to failure at 1°/s.

Histology

The explants were fixed by 10% zinc formalin for 24 h, decalcified in18% EDTA at 4° C. for 4 weeks, and embedded with glycol methacrylate andsectioned. The 3-μm sections were mounted onto slides for hematoxylin &eosin (H&E) staining.

Early-Stage In Vivo Cell Attachment on Implant Surfaces

To visualize the in vivo cell attachment to the hydrogel scaffoldsduring the early stage of guided bone regeneration, the hydrogelimplants with/without pre-loaded rhBMP-2 (500 ng/hydrogel) wereretrieved at 2 and 7 days post-op. The explants were fixed in 3.7%formaldehyde/DPBS solution, and the adherent cells were stained withAlexa Fluoro® 488 phalloidin (for F-actin staining, red) and DAPI (fornuclei staining, blue) following the vendor's protocol, respectively,and imaged on a Leica TCS SP2 confocal microscope. Phalloidin wasexcited at 495 nm and observed with a 518-nm filter while DAPI wasexcited at 368 nm and observed with a 461-nm filter.

In this specification and the appended claims, the singular forms “a,”“an,” and “the” include plural reference, unless the context clearlydictates otherwise.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Although any methods and materials similar or equivalent tothose described herein can also be used in the practice or testing ofthe present disclosure, the preferred methods and materials are nowdescribed. Methods recited herein may be carried out in any order thatis logically possible, in addition to a particular order disclosed.

Incorporation by Reference

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made in this disclosure. All such documents arehereby incorporated herein by reference in their entirety for allpurposes. Any material, or portion thereof, that is said to beincorporated by reference herein, but which conflicts with existingdefinitions, statements, or other disclosure material explicitly setforth herein is only incorporated to the extent that no conflict arisesbetween that incorporated material and the present disclosure material.In the event of a conflict, the conflict is to be resolved in favor ofthe present disclosure as the preferred disclosure.

Equivalents

The representative examples are intended to help illustrate theinvention, and are not intended to, nor should they be construed to,limit the scope of the invention. Indeed, various modifications of theinvention and many further embodiments thereof, in addition to thoseshown and described herein, will become apparent to those skilled in theart from the full contents of this document, including the examples andthe references to the scientific and patent literature included herein.The examples contain important additional information, exemplificationand guidance that can be adapted to the practice of this invention inits various embodiments and equivalents thereof.

What is claimed is: 1-53. (Canceled)
 54. An implant comprising a crosslinked 3-dimensioanl zwitterionic polymer and a protein, wherein the protein is encapsulated in the crosslinked 3-dimensioanl zwitterionic polymer and is characterized by preserved bioactivity as sequestered, wherein the crosslinked 3-dimensioanl zwitterionic polymer is polymethacrylate, polyacrylate, polymethacrylamide or polyacrylamide crosslinked with poly (ethylene glycol) dimethacrylate, poly(ethylene glycol) diacrylate, ethylene glycol diacrylate or ethylene glycol dimethacrylate.
 55. The implant of claim 54, wherein the protein is a therapeutic osteogenic protein.
 56. The implant of claim 54, wherein the protein is an anabolic agent.
 57. The implant of claim 54, wherein the protein is an angiogenic factor.
 58. The implant of claim 54, wherein the zwitterionic moiety comprises one or more selected from sulfobetaine, phosphorylcholine and carboxybetaine.
 59. The implant of claim 58, wherein the zwitterionic moiety is present in the polymer network as pendant groups to a polymeric backbone.
 60. The method of claim 59, wherein the zwitterionic moiety is present in the polymer at a density from about 0.05 mol% to about 10 mol%.
 61. The implant of claim 54, wherein the polymer is characterized by a crosslinking density, from about 0.05 mol% to about 10 mol%.
 62. The method of claim 54, wherein the implant is biodegradable.
 63. The implant of claim 54, suitable for implant as a dental implant or bone implant. 